In the study of the development of cells, fit flies have extensively been used as a model, as they are less complex than mammalian cells.
Pattern formation takes place through a series of logical steps, reiterated many times during the development of an organism. Viewed from a broader evolutionary perspective, across species, the same sort of reiterative pattern formations are seen. The central dogma of pattern formation has been described (Lawrence and Struhl, 1996). Three interlocking and overlapping steps are defined. Firstly, positional information in the form of morphogen gradients allocate cells into non-overlapping sets, each set founding a compartment. Secondly, each of these compartments acquire a genetic address, as a result of the function of active “selector” genes, that s cell fate within a compartment and also instruct cells and their descendents how to communicate with cells in neighboring compartments. The third step involves interactions between cells in adjacent compartments, initiating new morphogen gradients, which directly organize the pattern.
Taking these steps in greater detail, one finds the first step in patterning to be the definition of sets of cells in each primordium. Cells are allocated according to their positions with respect to both dorsoventral and anterior/posterior axes by morphogen gradients. Allocation of cells in the dorsoventral axis constitutes the germ layers, such as mesoderm or neurectoderm.
In segmentation, the second step (the specification of cell fate in each compartment) is carried out by the gene engrailed and elements of the bithorax complex. Engrailed defines anterior and posterior compartments both in segmentation and in limb specification.
The third step in pattern formation, secretion of morphogens, functions to differentiate patterns within compartments (and thereby establish segment polarity). Initially, all cells within a compartment are equipotent, but they become diversified to form pattern. Pattern formation depends on gradients of morphogens, gradients initiated along compartment boundaries. Such gradients are established by a short-range signal induced in all the cells of the compartment in which the above mentioned selector gene engrailed is active. For segment polarity, this signal is Hedgehog. In the adjacent compartment the selector gene is inactive, ensuring that the cells are sensitive to the signal. The Hedgehog signal range is probably only a few rows of cells wide; responding cells become a linear source of a long-range morphogen, that diffuses outward in all directions. There are three known Hedgehogs, Sonic (SHH), Indian (IHH) and Desert (DHH). The proteins they encode can substitute each for each other, but in wildtype animals, their distinct distributions result in unique activities. SHH controls the polarity of limb growth, directs the development of neurons in the ventral neural tube and patterns somities. IHH controls endochondral bone development and DHH is necessary for spermiogenesis. Vertebrate hedgehog genes are expressed in many other tissues, including the peripheral nervous system, brain, lung, liver, kidney, tooth primordia, genitalia and hindgut and foregut endoderm.
Thus, segment polarity genes have been identified in flies as mutations, which change the pattern of structures of the body segments. Mutations in these genes cause animals to develop the changed patterns on the surfaces of body segments, the changes affecting the pattern along the head to tail axis. For example, mutations in the gene patched cause each body segment to develop without the normal structures in the center of each segment. Instead there is a mirror image of the pattern normally found in the anterior segment. Thus, cells in the center of the segment make the wrong structures, and point them in the wrong direction with reference to the over all head-to-tail polarity of the animal.
About sixteen genes in the class are known. The encoded proteins include kinases, transcription factors, a cell junction protein, two secreted proteins called wingless (WG) and the above mentioned Hedgehog (HH), a single transmembrane protein called patched (PTC) and some novel proteins not related to any known protein. All of these proteins are beleived to work together in signaling pathways that inform cells about their neighbors in order to set cell fates and polarities.
PTC has been proposed as a receptor for HH protein based on genetic experiments in flies. A model for the relationship is that PTC acts through a largely unknown pathway to inactivate both its own transcription and the transcription of the wingless segment polarity gene. This model proposes that HH protein, secreted from adjacent cells, binds to the PTC receptor, inactivates it and thereby prevents PTC from turning off its own transcription or that of wingless. A number of experiments have shown coordinate events between PTC and HH.
Human patched gene (PTCH) was recently identified as the gene responsible for the nevoid basal cell carcinoma syndrome (NBCCS), also known as the Gorlin Syndrome, which is an autosomal dominant disorder that predisposes to both cancer and developmental defects (Gorlin (1995) Dermatologic Clinics 13:113-125) characterized by multiple basal cell carcinomas (BCCs), medulloblastomas and ovarian fibromas as well as numerous developmental anomalities (Hahn, H., Wicking, C., Zaphiropoulos, P. G., Gailani, M. R., Shanley, S., Chidambaram, A., Vorechovsky, I., Holmberg, E., Undén, A. B., Gillies, S., Negus, K., Smyth, I., Pressman, C., Leffell, D. J., Gerrard, B., Goldstein, A. M., Dean, M., Toftg{dot over (a)}rd, R., Chenevix-Trench, G., Wainright, B. and Bale, A. E. (1996): “Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome”, Cell 85, 841-851; and Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas, J. M., Quinn, A: G., Myers, R. M., Cox, D. R., Epstein, E. H. Jr and Scott, M. P. (1996): “Human homolog of patched, a candidate gene for the basal cell nevus syndrome”, Science 272, 1668-1671). PTCH codes for a membrane receptor of the autolytically cleaved (protein spliced), amino terminal domain of sonic hedgehog (SHH) (Mariago, V., Davey, R. A., Zuo, Y., Cunningham, J. M. and Tabin, C. J. (1996): “Biochemical evidence that patched is the Hedgehog receptor”, Nature 384, 176-179; and Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J. E., de Sauvage, F. and Rosenthal, A. (1996): “The tumor-suppressor gene patched encodes a candidate receptor for Sonic hedgehog”, Nature 384, 129-134). In the non-signalling state, PTCH is thought to inhibit the consecutive signalling of another membrane protein, smoothened (SMO), however binding of SHH to PTCH releives this inhibition (Goodrich, L. V., Milenkovic, L., Higgins, K. M. and Scott, M. P. (1997): “Altered neural cell fates and medullablastom in mouse patched mutants”, Science 277, 1109-1113). This cascade of signalling events, best characterized in Drsophila, also involves a number of intracellular components including fused (a serine threonine kinase), suppressor of fused, costal 2, and cubitus interruptus (Ruiz i Altaba, A.,: “Catching a Gli-mpse of Hedgehog” (1997) Cell 90, 193-196). The latter is a transcription factor that positively regulates the expression of target genes which also include PTCH itself.
Mutations in the PTCH gene have been identified in both sporadic and familial BCCs (Gailani, M. R., Stähle-Bäckdahl M., Leffell, D. J., Glynn, M., Zaphiropoulos, P. G., Pressman, C., Undén, A. B., Dean, M., Brash, D. E., Bale, A. E. and Toftg{dot over (a)}rd, R. (1996): “The role of human homologue of Drosophila patched in sporadic basal cell carcinomas” Nature Gene 14, 78-81). The lack of the normal PTCH protein in these cells allows the constitutive signalling of SMO to occur, resulting in the accumulation of mutant PTCH mRNAs (Undén, B. A., Zaphiropolous, P. G., Bruce, K., Toftg{dot over (a)}rd, R., and St{dot over (a)}hle-Bäckdahl M. (1997): “Human patched (PTCH) mRNA is overexpressed consistently in tumor cells of both familial and sporadic basal cell carcinoma”, Cancer Res. 57, 2336-2340). WO 96/11260 discloses the isolation of patched genes and the use of the PTC protein to identify ligands, other than the established ligand Hedgehog, that bind thereto.
However, there is still a need of a further understanding of the SHH/PTCH cell signalling, which may be provided by disclosure of further genes, peptides and proteins involved therein.